Non-alcoholic steatohepatitis (NASH) is a progressive disease of the liver of unknown ethiology characterized histologically by fatty acid accumulation, hepatocyte damage and inflammation resembling alcoholic hepatitis. NASH is a critical stage in the process that spans from hepatic steatosis to cirrhosis and liver failure. Obesity and type-2 diabetes are associated to NASH. Since the prevalence of these diseases is increasing, the prevalence of NASH is also expected to increase and therefore, this disease has become an emerging public issue in the United States [Reid A E. Nonalcoholic steatohepatitis. Gastroenterology 2001;121:710-723] as well as in other countries [Farell G C. Non-alcoholic steatohepatitis: what is it, and why is it important in the Asia-Pacific region? J Gastroenterol Hepatol 2003;18:124-138].
NASH is thought to arise from the interaction of many different genes and lifestyle factors. Mitochondrial impairment, oxidative stress and metabolic deregulation, have all been involved in the pathogenesis of steatohepatitis.
As it is true for other complex diseases, the genetic factors contributing to the development of NASH may be more readily identified by combining studies in patients with NASH and in animal models of the disease. One of these models is MAT1A knockout (MAT1A-KO) mice. These mice spontaneously develop NASH and hepatocellular carcinoma at about 8 and 15 months of age, respectively [Lu S C et al. Proc. Natl. Acad. Sci. USA 98:5560-5565 (2001); Martinez-Chantar M L et al. FASEB J 2002, 16(10), page 1292-1294. MAT1A gene encodes for methionine adenosyltransferase I and III, the main enzymes responsible of S-adenosylmethionine (SAMe) synthesis in liver. Earlier studies concluded that patients with liver cirrhosis and alcoholic hepatitis are deficient in SAMe synthesis; and that treatment with SAMe improves survival in patients with alcoholic liver cirrhosis.
Early diagnosis of NASH has been held back by the lack of reliable early markers of NASH development. Identification of genes and proteins expressed differentially in NASH with potential as biological markers or therapeutic targets could lead to the development of new tools for the diagnosis, prognosis and treatment of this disease.
A method for the diagnosis of NASH by using molecular markers based on the proteomic determination of a set of proteins detected in liver tissue samples has been disclosed (WO2004/055520).
Another method for the diagnosis of NASH based on the identification of a cluster of 85 discriminative early gene markers specific of NASH in liver tissue samples (thus, constituting what has been named the “genomic signature or fingerprint of NASH”) has been also described by the Applicant (EP 04103540.3). The technique used in that case combines liver samples from MAT1A-KO mice and from patients with early-stage NASH with bioinformatic and statistical methods to analyze the data generated from genome-wide expression profiling of the mentioned liver samples, thus allowing the identification of a cluster of discriminative early gene markers of steatohepatitis in mice and humans. Among the genes comprising the genomic signature of NASH there are enzymes (the majority being hydrolases, transferases and oxidoreductases), ligand-binding genes (heavy metal binding, nucleotide binding, protein binding, receptors and transcription factors); transporters (carbohydrate, electron transporter, and protein transporters); apoptosis regulators; chaperones; blood coagulation factors; and several genes with unknown function.
Looking for the presence of consensus sequences for vertebrate transcription factors among the promoters of the genes listed in Table 1 (EP 04103540.3), the inventors have now, surprisingly, found that only Sp1 transcription factor was present significantly in the promoters of a great number of the genes listed in said Table 1, more than would be expected by chance, suggesting that activation of Sp1 may be involved in the underlying mechanisms that lead to NASH.
Sp1 was one of the first eukaryotic transcription factors to be identified and cloned as a factor binding the SV40 early promoter (Dynan and Tjian, Cell 35:79-87, 1983). It is the founding memder of a family of proteins with highly homologous zinc-finger domains in the C-terminal region that bind GC or GT boxes, while the glutamine rich domains in the N-terminus are essential for transcriptional activation. Sp1 activates transcription by association with one of the co-activators associated with the TATA binding protein (TBP) in the TFIID complex. Other suggested roles for Sp1 in nuclear processes include remodeling of chromatin structures and maintenance of methylation-free CpG islands. Therefore, Sp1 is fundamental for the establishment of transcriptional competence, in addition to its role as a transcription factor. In a majority of promoters containing Sp1 binding sites, Sp1 provides a basal level of transcription. It plays an important role in the expression of numerous elements of the cell-cycle machinery, such as cyclins, Rb-like proteins, and E2F. Targeted disruption of the mouse Sp1 gene has shown that Sp1 is critical for normal embryogenesis. Sp1 (−/−) embryos are severely retarded in their development and display a marked heterogeneity in their phenotype. Interestingly, inactivation of the Sp1 gene is compatible with a certain degree of cell growth and differentiation, and the expression of various putative target genes, including that of certain cell cycle-related genes, was not altered in Sp1 (−/−) embryos. Also, CpG islands remained methylation free and active chromatin was formed at the globin loci. This may occur possibly because other members of the Sp1 family partially compensate for the absence of Sp1, thereby ameliorating the Sp1 knockout. Sp1 is involved in the basal expression of extracellular matrix (ECM) genes and is important in fibrotic processes. Thus, blocking Sp1 inhibits ECM gene expression what can be used in the treatment of fibrotic disorders (WO 02/066701).